Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Summary

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Abstract

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Introduction

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are widely exploited in regenerative medicine as well as experimental modeling of normal and diseased organogenesis because of their differentiation potential into cell lineages of all three germ layers^1,2. The differentiation fates of hPSCs are highly sensitive to local environmental factors that can modulate autocrine or paracrine signaling¹ as well as mechanotransduction processes mediated by physical cues^3-5. Cell micropatterning encompasses a set of techniques that have been developed to spatially organize the ....

Protocol

NOTE: This protocol describes the fabrication of PDMS stencil with 1,000 µm patterns by laser-cutting and micropatterning of the hESC line, H9 using the PDMS stencil.

1. Design and Fabrication of PDMS Stencil for Micropatterning

1. Design the stenciling sheet with through-holes of the desired geometry and size (e.g., 1,000 µm circles) and the stencil gasket using computer-aided design software¹⁰.
2. Laser-cut the stenciling sheet and gasket on 120-150 µm and 2 mm thick polydimethylsiloxane (PDMS) sheets respectively using a CO₂ laser-cutter¹⁰.
3. Bond the PDMS g....

Results

In this paper, we describe the fabrication of a cell stencil by using a laser cutter to generate 1,000 µm features. The stencil was composed of 2 parts: a thin stenciling sheet (approximately 100-200 µm thick) containing the micropattern through-holes, and a PDMS gasket to contain the ECM coating solution or cell suspension. Here, 127 µm and 2 mm thick commercially available PDMS sheets were used as the stenciling sheet and gasket respectively. Other methods for preparing PDMS sheets of c.......

Discussion

Fabrication of micropatterning stencils

Stencil micropatterning provides an ideal method to generate hPSC micropatterns for investigating niche-mediated differentiation patterning. The key advantage of stencil patterning over other micropatterning techniques, such as microcontact printing and photopatterning, is that it does not require surface modification and can be implemented on conventional TCPS substrates. Therefore, optimized culture media and ECM coatings for .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mm thick PDMS sheet	Specialty Silicone Products Inc., USA	SSPM823-.005	Used to form reservoir for stencil
120-150 μm thick PDMS sheet	Specialty Silicone Products Inc., USA	SSPM823-.040	Used to form stencil
60 mm Petri dish	Nunc Nunclon Delta	150326	Substrate for micropatterning
Accutase	Accutase, Merck Millipore, Singapore	SCR005	Enzyme to break H9 cells into single cells
Activin	R&D Systems, Singapore	338-AC-010	Growth factor for H9 differentiation
BMP4	R&D Systems, Singapore	338-BP-010	Growth factor for H9 differentiation
Plasma system	Femto Science, Korea	CUTE-MP	For plasma oxidation of stencil
Dispase	StemCell™ Technologies, Singapore	7923	Enzyme used to weaken the cell-ECM adhesion during passaging
DMEM/F12	GIBCO, USA	11330032	Basal medium for H9 cells
FGF2	R&D Systems, Singapore	233–FB–025	Growth factor for H9 differentiation
H9 cell line	WiCell Research Institute, Inc., USA	WA09	Human embryonic stem cells
hESC-qualified basement membrane matrix	Matrigel, BD Biosciences, Singapore	354277	Extra-cellular matrix coating to support growth of H9 cells
Inverted microscope	Leica Microsystems, Singapore	DMi1	For capturing bright-field images
Laser cutter	Epilog Helix 24 Laser System		Used to generate through holes in PDMS sheet
mTeSR™1 medium	StemCell™ Technologies, Singapore	5850	Maintainence medium for H9 cells
PDMS	SYLGARD® 184, Dow Corning Co., USA	3097358-1004	Used for sticking the PDMS stencil and reservior
ROCKi Y27632	Calbiochem, Merck Millipore, Singapore	688000	Maintains H9 cells as single cells
STEMdiff™ APEL™ medium	StemCell™ Technologies, Singapore	5210	Differentiation medium for H9 cells
Polyethylene terephthalate film	SureMark Singapore	SQ-6633	Used to form stencil
Cell culture compatible non-ionic surfactant	Pluronic acid F-127, Sigma, Singapore	P2443	Passivating reagent to repel cell adhesion in non-micropatterned substrates